青鳉低分子量激肽原(Kininogen)基因cDNA克隆与进化分析

Cloning and Evolutionary Analysis of Low Molecular Weight Kininogen Gene cDNA from Medaka (Oryzias latipes)

激肽原对半胱氨酸蛋白酶具有抑制作用,可结合血小板及内皮细胞,进而调控凝血、溶血以及调节血压等多种生理功能。本研究以青鳉(Oryzias latipes)为研究对象,通过RT-PCR和RACE(rapid amplification of cDNA ends)技术克隆了青鳉肝脏低分子量激肽原(LK)基因cDNA的全长序列(Gen Bank登录号:KP864678)。结果显示LK基因cDNA全长1 477 bp,其中5'端非翻译区190 bp,3'端非翻译区195 bp,开放性阅读框1 092 bp,编码363个氨基酸。青鳉LK蛋白属于分泌蛋白,N端含1个由21个氨基酸组成的信号肽;序列分析显示青鳉LK蛋白存在一个鱼类特有的保守蛋白酶抑制剂位点与1个缓激肽保守序列;Blastp同源性比对显示青鳉LK蛋白与其它鳉科鱼类的LK蛋白同源性均在67%以上,与哺乳动物LK蛋白同源性接近40%;同源建模显示青鳉LK蛋白存在2个空间上相对独立的半胱氨酸蛋白酶抑制剂样结构域。上述结果表明青鳉LK基因在进化过程中是保守的,可能发挥半胱氨酸蛋白酶抑制剂的作用。

Kininogen can inhibit the cysteine protease, and combine with endothelial cells and platelets to control blood coagulation and hemolysis and regulate blood pressure and other physiological functions. This research chose medaka Oryzias latipes as study object and cloned the full length cDNA of low molecular weight LK gene （GenBank accession number： FJ548753） from medaka liver by RT-PCR and RACE （rapid amplification of cDNA ends） methods. The results showed that full-length cDNA was 1 477 bp, which contained 190 bp 5＇-untranslated region, 195 bp 3＇-untranslated region and 1 092 bp open reading frame encoding 363 amino acids. Medaka LK protein belonged to secrete proteins, N-terminal contained a signal peptide composed of 21 amino acids; Sequence analysis showed that medaka LK protein contained a fish-specific conservative protease inhibitors site and a bradykinin conservative sequence; Blastp homology comparison showed that medaka LK protein homology was above 67% with other cyprinodontidae fish, and was nearly 40% with mammals; Homology modeling showed that there were two relatively spatial independent cysteine protease inhibitors-like domains in medaka LK protein. These results showed that medaka LK gene was conservative in the process of evolution and it might play the role of cysteine protease inhibitors.